1. Field of the Invention
The invention displays the fabrication method for a metal oxide nano-composite magnetic material and their uses in linkage, enrichment, and isolation of phosphorylated species. Furthermore, this invention especially addresses the applications for the enrichment and isolation of phosphorylated peptide/proteins from complex samples by using the proposed metal oxide nano-composite magnetic nanoparticles as affinity probes.
2. Description of Related Art
Protein phosphorylation is one of the most common protein post-translational modifications. Phosphorylated proteins play an important role for signal transduction and cell regulation in biological systems. Nevertheless, the abundance of phosphorylated proteins is generally low in biological samples (Cohen, P., Nature Cell Biology 4, E127-E130 (2002)). Therefore, it is necessary to develop suitable methods for selectively enriching traces of phosphorylated proteins from complex samples. Hence, many technologies for purifying phosphorylated proteins and phosphorylated peptides have been developed. Among these technologies, immobilized metal ion chromatography (IMAC), which immobilizes Fe3+ and Ga3+ onto solid substrates, has been used for purification of phosphorylated species from complex samples by Ficarro, S. B. et al., Nature Biotechnology 19, 301-305 (2002). The phosphorylated species can strongly interact with metal ions on the IMAC substrate.
Although IMAC has been widely used for purifications of phosphorylated proteins/peptides, there are some problems arising in IMAC. For example, non-specific binding, which may result in low sensitivity and poor extraction capacity, remains a problem in IMAC that has been investigated by Thomas, K. et al., J. Mass Spectrom. 38, 131-137 (2003). Metal oxide affinity chromatography (MOAC) is an alternative approach used for selectively enriching phosphorylated proteins/peptides based on the interaction between metal oxides and phosphates (Wolschin, F. et al., Proteomics 5, 4389-4397 (2005)). Conventionally, chromatographic columns packed with metal oxide coated beads are used for the enrichment of phosphorylated species. Columns packed with titanium oxide and zirconium oxide have been employed in MOAC for enriching phosphorylated peptides/proteins by Cantin, G. T. et al., Anal. Chem. 79, 4666-4673 (2007); Sugiyama N. et al., Mol. Cell Proteomics 6, 1103-1109 (2007). For ease of use, we have previously demonstrated that titanium oxide/zirconium coated magnetic nanoparticles can be used as effective affinity probes for phosphopeptides/proteins (Chen, C. T. and Chen, Y. C. Anal. Chem. 77, 5912-5919 (2005); Lo, C. Y.; Chen, W. Y.; Chen, C. T. and Chen, Y. C. J. Proteome Res. 6, 887-893 (2007)). This approach greatly simplifies the steps for isolation of probe target species from sample solutions because probe-target species conjugates can easily be isolated by magnetic separation. Moreover, the problem of non-specific binding is greatly reduced. We further explore several types of metal oxide coated magnetic nanoparticles (MOMNPs), which is the invention presented herein. In addition, microwave heating is utilized in accelerating the enrichment of phosphorylated proteins/peptides by MOMNPs because magnetic nanoparticles (MNPs) are good microwave absorbers, first investigated by Walkeiwicz, J. W. et al., Miner. Metall. Proc. 124, 247-252 (1988). Chen, W. Y. and Chen, Y. C. Anal. Chem. 79, 8061-8066 (2007), have previously demonstrated that extraction of traces of oligonucleotides from a sample solution could be completed using MNPs as the adsorbents under microwave heating within 30 sec. This invention also combines this technique to this MOMNPs approach. It has been demonstrated that metal oxide nano-composite magnetic material can be used to selectively enrich traces of phosphorylated species from complex samples within 30˜90 sec and then the target species of metal oxide nano-composite magnetic material can be characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Thus, the advantages of the present invention involve short analysis time, high specificity, and high sensitivity.